DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims Status
Claims 1-2, 4-11, 13-16, 18-21, and 24-26 are pending.
Claims 1-2, 5, 7-11, 13-16, 18, 20, and 24-26 are amended.
Election/Restrictions
Applicant’s election without traverse of Group 1 (claims 1, 2, 4-11, 13-16, 18-20, 24, and 25) in the reply filed on 05/06/2026 is acknowledged.
Applicant’s election without traverse of:
Species Group 1: BBE2.1
Species Group 2: solubility
Species Group 3: extracellular environment
Species Group 4: signal peptide
Species Group 5: SP4
Species Group 6: secreted protein
Species Group 7: lipase 2
Species Group 8: CNE1
Species Group 9: CNE1
Species Group 10: SP12
Species Group 11: enzymes involved in FAD biosynthesis
Species Group 12: native FAD synthetase
Species Group 13: FADS1
Species Group 14: YALI0F09163p
Species Group 15: modulated unfolded protein response
Species Group 16: CBGVA
Species Group 17: DVA
in the reply filed on 05/06/2026 is acknowledged.
Claim 26 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 05/06/2026.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 03/24/2026, 07/30/2024, and 07/17/2024 is acknowledged. The submission is in compliance with the provision of 37 CFR 1.97. Accordingly, the information disclosure statements have been considered.
Claim Objections
Claim 13 is objected to because of the following informalities:
Claim 13 is dependent on a rejected claim. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-2, 4-11, 13-16, 18-20, and 24-25 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 (and claims 4-11, 13-16, 18-20, and 24-25 dependent on) is directed to all possible cells expressing an exogenous terminal cannabinoid synthase “wherein the exogenous terminal cannabinoid synthase has at least one amino acid modification as compared to a wild-type exogenous terminal cannabinoid synthase”. The specification, however only provides representative species of a wild type exogenous terminal cannabinoid synthase. There is no disclosure of any particular structure to function/activity relationship in the disclosed species. The biosynthesis of cannabinoids remains incompletely understood at the molecular level (see Figure 2, Tahir et al., Journal of Cannabis Research, Vol. 3:7, published March 15, 2021; PMID: 33722296; also see Daniel et al., Archives of Biochemistry and Biophysics, Vol. 632., pg. 88-103; published October 15, 2017; also see Go et al., International Journal of Molecular Sciences, Vol. 24:1259; published 2023). The specification also fails to describe additional representative species of polypeptides with cannabinoid synthase activity by identifying structural characteristics or properties, for which no predictability of structure is apparent.
Regarding the level of skill and knowledge of the art of amino acid mutation, the reference of Singh et al. (Curr. Protein Pept. Sci. 18:1-11, 2017, cited on the attached Form PTO-892) reviews various protein engineering methods and discloses that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility, and conformational changes (see column 1, top, pg. 7). Also, the unpredictability associated with amino acid mutations is exemplified by the reference of Zhang et al. (Structure 26:1474-1485, 2018, cited on the attached Form PTO-892) which discloses that even a mutation of a surface residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide (column 1, pg. 1475).
Claim 2 is directed to all possible cells of claim 1, “wherein the exogenous terminal cannabinoid synthase is selected from i) a berberine bridge enzyme (BBE)-like family enzyme selected from the group consisting of BBE1.20, BBE1.21, BBE1.22, BBE2.6, BBE2.7, BBE2.8, BBE2.16, BBE2.18, BBE2.19, BBE2.20, BBE2.21, BBE2.22, and BBE2.14, having an amino acid sequence corresponding to SEQ ID NO: 19, 119, 120, 25-27, 121-126, and 33, respectively, ii) a berberine bridge enzyme-like enzyme comprising at least one amino acid modification compared to one of the amino acid sequences of BBE1.6 (SEQ ID NO: 118), BBE2.1 (SEQ ID NO: 20), BBE3.1 (SEQ ID NO: 34), BBE25.1 (SEQ ID NO: 59), BBE25.4 (SEQ ID NO: 62), and BBE25.5 (SEQ ID NO: 63), and iii) a functional fragment or derivative of any of the above having at least 70% sequence identity thereto”. The specification, however only provides representative species of a cell of claim 1, “wherein the exogenous terminal cannabinoid synthase is selected from i) a berberine bridge enzyme (BBE)-like family enzyme selected from the group consisting of BBE1.20, BBE1.21, BBE1.22, BBE2.6, BBE2.7, BBE2.8, BBE2.16, BBE2.18, BBE2.19, BBE2.20, BBE2.21, BBE2.22, and BBE2.14, having an amino acid sequence corresponding to SEQ ID NO: 19, 119, 120, 25-27, 121-126, and 33, respectively, and ii) a berberine bridge enzyme-like enzyme comprising amino acid the amino acid sequences of BBE1.6 (SEQ ID NO: 118), BBE2.1 (SEQ ID NO: 20), BBE3.1 (SEQ ID NO: 34), BBE25.1 (SEQ ID NO: 59), BBE25.4 (SEQ ID NO: 62), and BBE25.5 (SEQ ID NO: 63). There is no disclosure of any particular structure to function/activity relationship in the disclosed species. The biosynthesis of cannabinoids remains incompletely understood at the molecular level (see Figure 2, Tahir et al., Journal of Cannabis Research, Vol. 3:7, published March 15, 2021; PMID: 33722296; also see Daniel et al., Archives of Biochemistry and Biophysics, Vol. 632., pg. 88-103; published October 15, 2017; also see Go et al., International Journal of Molecular Sciences, Vol. 24:1259; published 2023). The specification also fails to describe additional representative species of polypeptides with cannabinoid synthase activity by identifying structural characteristics or properties, for which no predictability of structure is apparent.
Given this lack of additional representative species as encompassed by the claims, Applicants have failed to sufficiently describe the claimed invention, in such full, clear, concise, and exact terms that a skilled artisan would recognize Applicants were in possession of the claimed invention.
Applicant is referred to the revised guidelines concerning compliance with the written description requirement of U.S.C. 112, first paragraph, published in the Official Gazette and also available at www.uspto.gov.
Claim 1 (and claims 4-11, 13-16, 18-20, and 24-25 dependent on) is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a cell expressing a wild-type exogenous terminal cannabinoid synthase does not reasonably provide enablement for all possible cells expressing an exogenous terminal cannabinoid synthase “wherein the exogenous terminal cannabinoid synthase has at least one amino acid modification as compared to a wild-type exogenous terminal cannabinoid synthase”. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required, are summarized in In re Wands (858 F.2d 731, 8 USPQ 2nd 1400 (Fed. Cir. 1988)) as follows: (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claim(s).
Claim 1 (and claims 4-11, 13-16, 18-20, and 24-25 dependent on) is so broad as to encompass all possible cells expressing an exogenous terminal cannabinoid synthase “wherein the exogenous terminal cannabinoid synthase has at least one amino acid modification as compared to a wild-type exogenous terminal cannabinoid synthase”. The scope of the claims is not commensurate with the enablement provided by the disclosure with regard to the extremely large number of all possible cells expressing an exogenous terminal cannabinoid synthase “wherein the exogenous terminal cannabinoid synthase has at least one amino acid modification as compared to a wild-type exogenous terminal cannabinoid synthase” broadly encompassed by the claim. The claim rejected under this section of U.S.C. 112, first paragraph, places minimal structural limits on the required variant polypeptides having cannabinoid synthase activity encompassed by the claim. Since the amino acid sequence of a protein determines its structural and functional properties, predictability of which changes can be tolerated in a protein's amino acid sequence and obtain the desired activity requires a knowledge of and guidance with regard to which amino acids in the protein's sequence, if any, are tolerant of modification and which are conserved (i.e. expectedly intolerant to modification), and detailed knowledge of the ways in which the proteins' structure relates to its function. However, in this case the disclosure is limited to a cell expressing a wild-type exogenous terminal cannabinoid synthase.
While recombinant and mutagenesis techniques are known, it is not routine in the art to screen for multiple substitutions or multiple modifications, as encompassed by the instant claims, and the positions within a protein's sequence where amino acid modifications can be made with a reasonable expectation of success in obtaining the desired activity/utility are limited in any protein and the result of such modifications is unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given protein to diminish with each further and additional modification, e.g. multiple substitutions.
The specification does not support the broad scope of the claims which encompass any possible cells of claim 1, expressing an exogenous terminal cannabinoid synthase “wherein the exogenous terminal cannabinoid synthase has at least one amino acid modification as compared to a wild-type exogenous terminal cannabinoid synthase” because the specification does not establish: (A) regions of the polypeptide which may be modified effecting the cannabinoid synthase; (B) the general tolerance of enzymes of the cannabinoid synthase group to modification and extent of such tolerance; (C) a rational and predictable scheme for modifying any amino acid residue of an enzyme of the cannabinoid synthase group with an expectation of obtaining the desired biological function; and (D) the specification provides insufficient guidance as to which of the essentially infinite possible choices is likely to be successful. Because of this lack of guidance, the extended experimentation that would be required to determine which substitutions would be acceptable to retain the required terminal cannabinoid synthase activities and the fact that the relationship between the sequence of a peptide and its tertiary structure (i.e. its activity) are not well understood and are not predictable (e.g., see Sheng et al, FEBS J., Vol. 24, pg. 4703-13, 2015, Ngo et al. in The Protein Folding Problem and Tertiary Structure Prediction, 1994, Merz et al. (ed.), Birkhauser, Boston, MA, pp. 433 and 492-495; Franceus et al., J. Ind. Microbiol. Biotechnol. Vol 44, pp 687-695, 2017), it would require undue experimentation for one skilled in the art to arrive at the majority of polypeptides having cannabinoid synthase enzymatic activity of the claimed genus.
.
Claim 2 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a “cell of claim 1, wherein the exogenous terminal cannabinoid synthase is selected from i) a berberine bridge enzyme (BBE)-like family enzyme selected from the group consisting of BBE1.20, BBE1.21, BBE1.22, BBE2.6, BBE2.7, BBE2.8, BBE2.16, BBE2.18, BBE2.19, BBE2.20, BBE2.21, BBE2.22, and BBE2.14, having an amino acid sequence corresponding to SEQ ID NO: 19, 119, 120, 25-27, 121-126, and 33, respectively, and ii) a berberine bridge enzyme-like enzyme comprising amino acid the amino acid sequences of BBE1.6 (SEQ ID NO: 118), BBE2.1 (SEQ ID NO: 20), BBE3.1 (SEQ ID NO: 34), BBE25.1 (SEQ ID NO: 59), BBE25.4 (SEQ ID NO: 62), and BBE25.5 (SEQ ID NO: 63)” , does not reasonably provide enablement for all possible “cell of claim 1, wherein the exogenous terminal cannabinoid synthase is selected from i) a berberine bridge enzyme (BBE)-like family enzyme selected from the group consisting of BBE1.20, BBE1.21, BBE1.22, BBE2.6, BBE2.7, BBE2.8, BBE2.16, BBE2.18, BBE2.19, BBE2.20, BBE2.21, BBE2.22, and BBE2.14, having an amino acid sequence corresponding to SEQ ID NO: 19, 119, 120, 25-27, 121-126, and 33, respectively, ii) a berberine bridge enzyme-like enzyme comprising at least one amino acid modification compared to one of the amino acid sequences of BBE1.6 (SEQ ID NO: 118), BBE2.1 (SEQ ID NO: 20), BBE3.1 (SEQ ID NO: 34), BBE25.1 (SEQ ID NO: 59), BBE25.4 (SEQ ID NO: 62), and BBE25.5 (SEQ ID NO: 63), and iii) a functional fragment or derivative of any of the above having at least 70% sequence identity thereto”. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required, are summarized in In re Wands (858 F.2d 731, 8 USPQ 2nd 1400 (Fed. Cir. 1988)) as follows: (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claim(s).
Claim 2 is so broad as to encompass all possible cells of claim 1, “wherein the exogenous terminal cannabinoid synthase is selected from i) a berberine bridge enzyme (BBE)-like family enzyme selected from the group consisting of BBE1.20, BBE1.21, BBE1.22, BBE2.6, BBE2.7, BBE2.8, BBE2.16, BBE2.18, BBE2.19, BBE2.20, BBE2.21, BBE2.22, and BBE2.14, having an amino acid sequence corresponding to SEQ ID NO: 19, 119, 120, 25-27, 121-126, and 33, respectively, ii) a berberine bridge enzyme-like enzyme comprising at least one amino acid modification compared to one of the amino acid sequences of BBE1.6 (SEQ ID NO: 118), BBE2.1 (SEQ ID NO: 20), BBE3.1 (SEQ ID NO: 34), BBE25.1 (SEQ ID NO: 59), BBE25.4 (SEQ ID NO: 62), and BBE25.5 (SEQ ID NO: 63), and iii) a functional fragment or derivative of any of the above having at least 70% sequence identity thereto”. The scope of the claims is not commensurate with the enablement provided by the disclosure with regard to the extremely large number of cells of claim 1, “wherein the exogenous terminal cannabinoid synthase is selected from i) a berberine bridge enzyme (BBE)-like family enzyme selected from the group consisting of BBE1.20, BBE1.21, BBE1.22, BBE2.6, BBE2.7, BBE2.8, BBE2.16, BBE2.18, BBE2.19, BBE2.20, BBE2.21, BBE2.22, and BBE2.14, having an amino acid sequence corresponding to SEQ ID NO: 19, 119, 120, 25-27, 121-126, and 33, respectively, ii) a berberine bridge enzyme-like enzyme comprising at least one amino acid modification compared to one of the amino acid sequences of BBE1.6 (SEQ ID NO: 118), BBE2.1 (SEQ ID NO: 20), BBE3.1 (SEQ ID NO: 34), BBE25.1 (SEQ ID NO: 59), BBE25.4 (SEQ ID NO: 62), and BBE25.5 (SEQ ID NO: 63), and iii) a functional fragment or derivative of any of the above having at least 70% sequence identity thereto” broadly encompassed by the claim. The claim rejected under this section of U.S.C. 112, first paragraph, places minimal structural limits on the required variant polypeptides having cannabinoid synthase activity encompassed by the claim. Since the amino acid sequence of a protein determines its structural and functional properties, predictability of which changes can be tolerated in a protein's amino acid sequence and obtain the desired activity requires a knowledge of and guidance with regard to which amino acids in the protein's sequence, if any, are tolerant of modification and which are conserved (i.e. expectedly intolerant to modification), and detailed knowledge of the ways in which the proteins' structure relates to its function. However, in this case the disclosure is limited to “cell of claim 1, wherein the exogenous terminal cannabinoid synthase is selected from i) a berberine bridge enzyme (BBE)-like family enzyme selected from the group consisting of BBE1.20, BBE1.21, BBE1.22, BBE2.6, BBE2.7, BBE2.8, BBE2.16, BBE2.18, BBE2.19, BBE2.20, BBE2.21, BBE2.22, and BBE2.14, having an amino acid sequence corresponding to SEQ ID NO: 19, 119, 120, 25-27, 121-126, and 33, respectively, and ii) a berberine bridge enzyme-like enzyme comprising amino acid the amino acid sequences of BBE1.6 (SEQ ID NO: 118), BBE2.1 (SEQ ID NO: 20), BBE3.1 (SEQ ID NO: 34), BBE25.1 (SEQ ID NO: 59), BBE25.4 (SEQ ID NO: 62), and BBE25.5 (SEQ ID NO: 63)”.
While recombinant and mutagenesis techniques are known, it is not routine in the art to screen for multiple substitutions or multiple modifications, as encompassed by the instant claims, and the positions within a protein's sequence where amino acid modifications can be made with a reasonable expectation of success in obtaining the desired activity/utility are limited in any protein and the result of such modifications is unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given protein to diminish with each further and additional modification, e.g. multiple substitutions.
The specification does not support the broad scope of the claims which encompass any possible cells of claim 1, “wherein the exogenous terminal cannabinoid synthase is selected from i) a berberine bridge enzyme (BBE)-like family enzyme selected from the group consisting of BBE1.20, BBE1.21, BBE1.22, BBE2.6, BBE2.7, BBE2.8, BBE2.16, BBE2.18, BBE2.19, BBE2.20, BBE2.21, BBE2.22, and BBE2.14, having an amino acid sequence corresponding to SEQ ID NO: 19, 119, 120, 25-27, 121-126, and 33, respectively, ii) a berberine bridge enzyme-like enzyme comprising at least one amino acid modification compared to one of the amino acid sequences of BBE1.6 (SEQ ID NO: 118), BBE2.1 (SEQ ID NO: 20), BBE3.1 (SEQ ID NO: 34), BBE25.1 (SEQ ID NO: 59), BBE25.4 (SEQ ID NO: 62), and BBE25.5 (SEQ ID NO: 63), and iii) a functional fragment or derivative of any of the above having at least 70% sequence identity thereto” because the specification does not establish: (A) regions of the polypeptide which may be modified effecting the cannabinoid synthase; (B) the general tolerance of enzymes of the cannabinoid synthase group to modification and extent of such tolerance; (C) a rational and predictable scheme for modifying any amino acid residue of an enzyme of the cannabinoid synthase group with an expectation of obtaining the desired biological function; and (D) the specification provides insufficient guidance as to which of the essentially infinite possible choices is likely to be successful. Because of this lack of guidance, the extended experimentation that would be required to determine which substitutions would be acceptable to retain the required terminal cannabinoid synthase activities and the fact that the relationship between the sequence of a peptide and its tertiary structure (i.e. its activity) are not well understood and are not predictable (e.g., see Sheng et al, FEBS J., Vol. 24, pg. 4703-13, 2015, Ngo et al. in The Protein Folding Problem and Tertiary Structure Prediction, 1994, Merz et al. (ed.), Birkhauser, Boston, MA, pp. 433 and 492-495; Franceus et al., J. Ind. Microbiol. Biotechnol. Vol 44, pp 687-695, 2017), it would require undue experimentation for one skilled in the art to arrive at the majority of polypeptides having cannabinoid synthase enzymatic activity of the claimed genus.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-2, 4-6, 8, 10-11, 14-15, 20, and 24 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020), hereinafter referred to as Demetrix.
With regards to claim 1, Demetrix discloses modified host cells comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide (see paragraph 0085, pg. 13) including cannabinoid synthase polypeptides such as tetrahydrocannabinolic acid (THCA) synthase polypeptides (see paragraph 0087, pg. 13-14). Demetrix further discloses that the modified host cells comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperones or co-chaperone polypeptides (overexpression) (see Paragraph 009, pg. 3). Demetrix discloses that a cannabinoid synthase polypeptide may be a variant of a THCAS polypeptide (see Paragraph 00276, pg. 67) and the polypeptides may be variants differing from a wild-type polypeptide by amino acid mutations (see Paragraph 00105, pg. 19). Demetrix also discloses that suitable host cells may include Saccharomyces cerevisiae or Yarrowia lipolytica (see Paragraphs 00429-00430, pg. 143-144).
With regards to claim 2, Demetrix discloses that heterologous nucleic acids comprise a nucleotide sequence encoding a BBE or BBE-like polypeptide (see Paragraph 0008, pg. 2) and an embodiment can include a tetrahydrocannabinolic acid synthase polypeptide according to SEQ ID NO: 91 (see Paragraph 00467, pg. 159). SEQ ID NO: 91 has at least one amino acid modification compared to BBE2.1 (SEQ ID NO: 20) of the current instant application (see alignment below).
Title: US-18-701-596-20
Perfect score: 2773
Sequence: 1 NPRENFLKCFSKHIPNNVAN..........DPNNFFRNEQSIPPLPPHHH 517
Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5
Searched: 1 seqs, 369 residues
Total number of hits satisfying chosen parameters: 1
Minimum DB seq length: 0
Maximum DB seq length: inf
Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 50 summaries
Database : AASEQ2_05122026_115420.fasta:*
SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 1707 61.6 369 1 AASEQ2_05122026_115420
ALIGNMENTS
RESULT 1
AASEQ2_05122026_115420
Query Match 61.6%; Score 1707; DB 1; Length 369;
Best Local Similarity 95.8%;
Matches 323; Conservative 4; Mismatches 8; Indels 2; Gaps 1;
Qy 1 NPRENFLKCFSKHIPNNVANPKLVYTQHDQLYMSILNSTIQNLRFISDTTPKPLVIVTPS 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 29 NPRENFLKCFSKHIPNNVANPKLVYTQHDQLYMSILNSTIQNLRFISDTTPKPLVIVTPS 88
Qy 61 NNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTAW 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 89 NNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTAW 148
Qy 121 VEAGATLGEVYYWINEKNENLSFPGGYCPTVGVGGHFSGGGYGALMRNYGLAADNIIDAH 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 149 VEAGATLGEVYYWINEKNENLSFPGGYCPTVGVGGHFSGGGYGALMRNYGLAADNIIDAH 208
Qy 181 LVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWKIKLVAVPSKSTIFSVKKNMEIHG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 209 LVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWKIKLVAVPSKSTIFSVKKNMEIHG 268
Qy 241 LVKLFNKWQNIAYKYDKDLVLMTHFITKNITDNHGKNKTTVHGYFSSIFHGGVDSLVDLM 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 269 LVKLFNKWQNIAYKYDKDLVLMTHFITKNITDNHGKNKTTVHGYFSSIFHGGVDSLVDLM 328
Qy 301 NKSFPELGIKKTDCKEFSWI--DTTIFYSGVVNFNTA 335
|||||||||||||||||||| ::| | : |:
Db 329 NKSFPELGIKKTDCKEFSWIVLPSSILVSSISTLLTS 365
With regards to claim 4, Demetrix teaches that modification of the secretory pathway in the host cell may improve expression and solubilization of BBE or BBE-like polypeptides (see Paragraph 009, pg. 15).
With regards to claims 5-6, Demetrix teaches that the cannabinoid synthase polypeptides contain an N-terminal secretory signal sequence and that the signal sequence is thought to mediate routing through the secretory pathway where the enzyme is matured (see Paragraph 0087, pg. 13-14).
With regards to claim 8, that the nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide comprises a nucleotide sequence encoding a GFP polypeptide and that after transcription and translation, the resulting BBE or BBE-like polypeptide is fused to the GFP polypeptide (secreted protein) (see Paragraph 00488, pg. 168-169). Demetrix further discloses a CBDAS polypeptide fused to a GFP polypeptide via a non-cleavable flexible linker. Demetrix discloses that the resulting fusion protein can be used to examine how engineering the secretory pathway affects synthase functional expression (see paragraph 00614, pg. 242).
With regards to claim 10-11, Demetrix discloses that the modified host cells comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone polypeptides such as a CNE1 polypeptide (see Paragraph 00131, pg. 28).
With regards to claim 14-15, Demetrix discloses that the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides (see Paragraph 0009, pg. 3). Saccharomyces cerevisiae inherently encodes a native FAD synthetase as evidenced by Uniprot database entry (FAD1_yeast; February 1, 1995) and Wu et al. (Molecular and Cellular Biology, Vol. 15., pg. 264-271, published January 1995) who disclose the cloning and characterization of FAD1 of Saccharomyces cerevisiae (see Abstract, pg. 264).
With regards to claim 20, Demetrix discloses that the modified host cell comprises a deletion of one or more genes such as ROT2 (see Paragraph 00125, pg. 26). Demetrix defines ROT2 as a glucosidase (see Paragraph 0099, pg. 17).
With regards to claim 24, Demetrix discloses that modified host cells may comprise one or more modifications to modulate the expression of one ore more polypeptides involved in the unfolded protein response (see Paragraph 00215, pg. 50). `
Therefore, claims 1-2, 4-6, 8, 10-12, 14-15, 20, and 24 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020).
Claims 1, 4, and 25 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Genomatica, Inc. (WIPO International Publication Number WO 2021/211611 A1; published October 21, 2021, International filing date April 13, 2021), hereinafter referred to as Genomatica.
With regards to claim 1, Genomatica discloses an engineered cell for making non-natural THCAS (terminal cannabinoid synthases) (see description). Genomatica discloses that the non-natural cannabinoid synthase comprises at least one amino acid variation as compared to the wild-type cannabinoid synthase THCAS (see Abstract). Genomatica further discloses that the cells may have a second plasmid that express chaperones (overexpression) to help facilitate correct protein folding of THCAS (see paragraph 00438, pg. 155). In particular, Genomatica discloses that the chaperone expression plasmid pGro7 may be in the E. coli cells along with the plasmid that expresses the THCAS (see paragraph 00438, pg. 155).
With regards to claim 4, Genomatica teaches that in some embodiments, the non-natural THCAS comprising a salt bridge and no disulfide bond between aA and aC has improved solubility (see Paragraph 0156, pg. 45).
With regards to claim 25, Genomatica discloses that in some embodiments, the engineered cell comprises a prenyltransferase (see Paragraph 0053, pg. 17) that can catalyze conversion of olivetolic acid (OA) to CGBA (see Paragraph 0070, pg. 22) and in some embodiments provides a method for making CBGVA (see Paragraph 0056, pg. 20).
Therefore, claims 1, 4, and 25 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Genomatica, Inc. (WIPO International Publication Number WO 2021/211611 A1; published October 21, 2021, International filing date April 13, 2021), hereinafter referred to as Genomatica.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 2, 4-6, 8, 10-11, 14-15, 20 and 24 are rejected under pre-AIA 35 U.S.C. 102 (a)(1) and 102(a)2 as anticipated by or, in the alternative, under pre-AIA 35 U.S.C. 103(a) as obvious over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020), hereinafter referred to as Demetrix.
With regards to claim 1, Demetrix teaches modified host cells comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide (see paragraph 0085, pg. 13) including cannabinoid synthase polypeptides such as tetrahydrocannabinolic acid (THCA) synthase polypeptides (see paragraph 0087, pg. 13-14). Demetrix further teaches that the modified host cells comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperones or co-chaperone polypeptides (overexpression) (see Paragraph 009, pg. 3). Demetrix teaches that a cannabinoid synthase polypeptide may be a variant of a THCAS polypeptide (see Paragraph 00276, pg. 67) and the polypeptides may be variants differing from a wild-type polypeptide by amino acid mutations (see Paragraph 00105, pg. 19). Demetrix also teaches that suitable host cells may include Saccharomyces cerevisiae or Yarrowia lipolytica (see Paragraphs 00429-00430, pg. 143-144).
With regards to claim 2, Demetrix teaches that heterologous nucleic acids comprise a nucleotide sequence encoding a BBE or BBE-like polypeptide (see Paragraph 0008, pg. 2) and an embodiment can include a tetrahydrocannabinolic acid synthase polypeptide according to SEQ ID NO: 91 (see Paragraph 00467, pg. 159). SEQ ID NO: 91 has at least one amino acid modification compared to BBE2.1 (SEQ ID NO: 20) of the current instant application (see alignment below).
Title: US-18-701-596-20
Perfect score: 2773
Sequence: 1 NPRENFLKCFSKHIPNNVAN..........DPNNFFRNEQSIPPLPPHHH 517
Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5
Searched: 1 seqs, 369 residues
Total number of hits satisfying chosen parameters: 1
Minimum DB seq length: 0
Maximum DB seq length: inf
Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 50 summaries
Database : AASEQ2_05122026_115420.fasta:*
SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 1707 61.6 369 1 AASEQ2_05122026_115420
ALIGNMENTS
RESULT 1
AASEQ2_05122026_115420
Query Match 61.6%; Score 1707; DB 1; Length 369;
Best Local Similarity 95.8%;
Matches 323; Conservative 4; Mismatches 8; Indels 2; Gaps 1;
Qy 1 NPRENFLKCFSKHIPNNVANPKLVYTQHDQLYMSILNSTIQNLRFISDTTPKPLVIVTPS 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 29 NPRENFLKCFSKHIPNNVANPKLVYTQHDQLYMSILNSTIQNLRFISDTTPKPLVIVTPS 88
Qy 61 NNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTAW 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 89 NNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTAW 148
Qy 121 VEAGATLGEVYYWINEKNENLSFPGGYCPTVGVGGHFSGGGYGALMRNYGLAADNIIDAH 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 149 VEAGATLGEVYYWINEKNENLSFPGGYCPTVGVGGHFSGGGYGALMRNYGLAADNIIDAH 208
Qy 181 LVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWKIKLVAVPSKSTIFSVKKNMEIHG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 209 LVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWKIKLVAVPSKSTIFSVKKNMEIHG 268
Qy 241 LVKLFNKWQNIAYKYDKDLVLMTHFITKNITDNHGKNKTTVHGYFSSIFHGGVDSLVDLM 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 269 LVKLFNKWQNIAYKYDKDLVLMTHFITKNITDNHGKNKTTVHGYFSSIFHGGVDSLVDLM 328
Qy 301 NKSFPELGIKKTDCKEFSWI--DTTIFYSGVVNFNTA 335
|||||||||||||||||||| ::| | : |:
Db 329 NKSFPELGIKKTDCKEFSWIVLPSSILVSSISTLLTS 365
With regards to claim 4, Demetrix teaches that modification of the secretory pathway in the host cell may improve expression and solubilization of BBE or BBE-like polypeptides (see Paragraph 009, pg. 15).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the current instant applications to incorporate the above features in claims 1-4 into the modified host cell to overexpress a cannabinoid synthase harboring one or more mutations in the exogenous terminal cannabinoid synthase as compared to a wild-type exogenous terminal cannabinoid synthase as well as overexpressing one or more chaperones. It would have also been obvious to one of ordinary skill in the art to make modifications to the secretory pathway or system in the host cell as taught by Demetrix (see Paragraph 0093, pg. 15) since this may improve expression and solubilization of BBE-like polypeptides, since these polypeptides are often processed in the secretory pathway. One would be motivated to do so as taught by Demetrix to achieve expression of the exogenous cannabinoid synthase as well as chaperones that can assist in protein folding and translocation. One of ordinary skill in the art of protein engineering would have expectations of success in doing so as Demetrix teaches all the methods and protocols needed to do so.
With regards to claims 5-6, Demetrix teaches that the cannabinoid synthase polypeptides contain an N-terminal secretory signal sequence and that the signal sequence is thought to mediate routing through the secretory pathway where the enzyme is matured (see Paragraph 0087, pg. 13-14).
With regards to claim 8, Demetrix teaches that the nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide comprises a nucleotide sequence encoding a GFP polypeptide and that after transcription and translation, the resulting BBE or BBE-like polypeptide is fused to the GFP polypeptide (secreted protein) (see Paragraph 00488, pg. 168-169). Demetrix further teaches a CBDAS polypeptide fused to a GFP polypeptide via a non-cleavable flexible linker. Demetrix teaches that the resulting fusion protein can be used to examine how engineering the secretory pathway affects synthase functional expression (see paragraph 00614, pg. 242).
With regards to claim 10-11, Demetrix teaches that the modified host cells comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone polypeptides such as a CNE1 polypeptide (see Paragraph 00131, pg. 28).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the current instant application to incorporate the above features taught by Demetrix with regards to claims 5-6, 8, and 10-11 into the host cell taught by Demetrix. One of ordinary skill in the art of protein engineering would be motivated to do so since incorporating a signal sequence or fusing to a secreted protein to the cannabinoid synthase would aide in secretion and/or localization of the expressed cannabinoid synthase polypeptide. Modification of the host cell by incorporating expression of a chaperone CNE1 would provide the benefit of providing protein quality control. One of ordinary skill in the art of protein engineering would have expectations of success since Demetrix provides the necessary teachings to do so.
With regards to claim 14-15, Demetrix teaches that the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides (see Paragraph 0009, pg. 3). Saccharomyces cerevisiae inherently encodes a native FAD synthetase as evidenced by Uniprot database entry (FAD1_yeast; February 1, 1995) and Wu et al. (Molecular and Cellular Biology, Vol. 15., pg. 264-271, published January 1995) who disclose the cloning and characterization of FAD1 of Saccharomyces cerevisiae (see Abstract, pg. 264).
With regards to claim 20, Demetrix teaches that the modified host cell comprises a deletion of one or more genes such as ROT2 (see Paragraph 00125, pg. 26). Demetrix defines ROT2 as a glucosidase (see Paragraph 0099, pg. 17).
With regards to claim 24, Demetrix teaches that modified host cells may comprise one or more modifications to modulate the expression of one more polypeptides involved in the unfolded protein response (see Paragraph 00215, pg. 50).
It would have been obvious to one of ordinary skill in the art of protein engineering before the effective filing date of the current instant application to incorporate the teachings of Demetrix as applied to claims 14-15, 20, and 24 into the host cell of claim 1. One of ordinary skill in the art would be motivated to do so since modification of the host cell with an exogenous FAD synthetase would provide biosynthesis of the FAD cofactor. Deletion of ROT2 to inactivate ROT2 glucosidase and modulation of the unfolded protein response as taught by Demetrix would aide in protein quality control. One of ordinary skill in the art would have expectations of success in doing so as Demetrix teaches all the necessary methods to do so.
Therefore, claims 1, 2, 4-6, 8, 10-11, 14-15, 20 and 24 are rejected under pre-AIA 35 U.S.C. 102 (a)(1) and 102(a)2 as anticipated by or, in the alternative, under pre-AIA 35 U.S.C. 103(a) as obvious over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020).
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020) as applied to claims 1, 5, and 6 above, and further in view of Keasling et al. (US Patent No: US 10,563,211 B2; published February 18, 2020), hereinafter referred to as Keasling.
The teachings of Demetrix as applied to claims 1, 5, and 6 are summarized above.
Demetrix does not teach that the cannabinoid synthase is expressed with a signal peptide selected from SP11.
However, Keasling teaches a THCA synthase (cannabinoid synthase) that was introduced into a strain with polypeptide tags such as the ProA signal sequence (MIFDGTTMSIAIGLLSTLGIGAEA) (see Column 333, lines 40-47). The sequence of the ProA signal sequence is 100% identical to the SP11 sequence (SEQ ID NO: 96) of the current instant application.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the current instant application to use the ProA signal sequence as an option or expressing the cannabinoid synthase with a signal sequence. One of ordinary skill in the art of cloning and protein purification would be motivated to do so based on the teachings of Keasling who demonstrated the successful use of ProA signal sequence as an attached tag to a similar cannabinoid synthase, THCA in achieving enhanced protein expression and secretion. One of ordinary skill in the art of cloning and protein production would have high expectations of success as the combined teachings of Demetrix and Keasling provide the necessary teachings and protocols.
Therefore, claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020) as applied to claims 1, 5, and 6 above, and further in view of Keasling et al. (US Patent No: US 10,563,211 B2; published February 18, 2020).
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020) as applied to claims 1 and 8 above, and further in view of Hofmeyer et al. (Molecular Biotechnology, Vol. 56., pg. 79-90; published 2014), hereinafter referred to as Hofmeyer and further in view of Uniprot Database entry (F2Z685_YARLI; May31, 20011 entry version).
The teaching of Demetrix as applied to claims 1 and 8 are summarized above.
Demetrix does not teach that the cannabinoid synthase is fused to a lipase 2 (Lip2) having the amino acid sequence of SEQ ID NO: 100.
However, Hofmeyer teaches a strategy for protein production and purification via expression in Yarrowia lipolytica as a Lip2p fusion protein. Hofmeyer teaches that expression of proteins of interest via Lip2p fusion not only provides a convenient expression and purification scheme but also enables for online monitoring of accumulation of secreted fusion proteins in the medium (see Abstract pg. 79). Hofmeyer further teaches that the fusion protein system using Y. lipolytica as host organism and Lip2p as a carrier for secretion and export allows for the effective expression and purification of proteins of different sizes and functionalities (see Conclusions, pg. 88).
Hofmeyer is silent on the Lip2p having the sequence of SEQ ID NO: 100 of the current instant application.
However, Uniprot Database entry F2Z685_YARLI teaches a triacylglycerol lipase from Yarrowia lipolytica having 100% sequence identity with SEQ ID NO: 100 of the current instant application (see alignment below).
Title: US-18-701-596-100
Perfect score: 1782
Sequence: 1 MKLSTILFTACATLAAALPS..........QVNVIGNHLQYFVTEGVCGI 334
Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5
Searched: 1 seqs, 334 residues
Total number of hits satisfying chosen parameters: 1
Minimum DB seq length: 0
Maximum DB seq length: inf
Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 50 summaries
Database : AASEQ2_05132026_121715.fasta:*
SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 1782 100.0 334 1 AASEQ2_05132026_121715
ALIGNMENTS
RESULT 1
AASEQ2_05132026_121715
Query Match 100.0%; Score 1782; DB 1; Length 334;
Best Local Similarity 100.0%;
Matches 334; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MKLSTILFTACATLAAALPSPITPSEAAVLQKRVYTSTETSHIDQESYNFFEKYARLANI 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MKLSTILFTACATLAAALPSPITPSEAAVLQKRVYTSTETSHIDQESYNFFEKYARLANI 60
Qy 61 GYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFHDPRLIFDVSGYLAVDHASKQIYLVIRG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 GYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFHDPRLIFDVSGYLAVDHASKQIYLVIRG 120
Qy 121 THSLEDVITDIRIMQAPLTNFDLAANISSTATCDDCLVHNGFIQSYNNTYNQIGPKLDSV 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 THSLEDVITDIRIMQAPLTNFDLAANISSTATCDDCLVHNGFIQSYNNTYNQIGPKLDSV 180
Qy 181 IEQYPDYQIAVTGHSLGGAAALLFGINLKVNGHDPLVVTLGQPIVGNAGFANWVDKLFFG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 IEQYPDYQIAVTGHSLGGAAALLFGINLKVNGHDPLVVTLGQPIVGNAGFANWVDKLFFG 240
Qy 241 QENPDVSKVSKDRKLYRITHRGDIVPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 QENPDVSKVSKDRKLYRITHRGDIVPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQG 300
Qy 301 QSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI 334
||||||||||||||||||||||||||||||||||
Db 301 QSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI 334
It would have therefore been obvious to one of ordinary skill in the art before the effective filing date of the current instant application to fuse the cannabinoid synthase taught by Demetrix to a lipase 2 as taught by Hofmeyer and Uniprot Database entry F2Z685_YARLI. One of ordinary skill in the art of protein expression would be motivated to do so since Hofmeyer teaches the fusion protein system using Lip2p as a carrier for secretion and export allows for the effective expression and purification of proteins of different sizes and functionalities. Fusion of the cannabinoid synthase taught by Demetrix to the lipase 2 as taught by Hofmeyer and Uniprot Database entry F2Z685 would also provide secretion and export activity to the fusion protein. One of ordinary skill in the art of protein engineering would have high expectations of success in doing so since Demetrix, Hofmeyer, and Uniprot Database entry F2Z685_YALI teach all the methods and protocols needed.
Therefore, claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020) as applied to claims 1 and 8 above, and further in view of Hofmeyer et al. (Molecular Biotechnology, Vol. 56., pg. 79-90; published 2014) and further in view of Uniprot Database entry (F2Z685_YARLI; May31, 20011 entry version).
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020) as applied to claims 1, 14, and 15 above, and further in view of Uniprot Database entry (Q6C7T3; August 16, 2004).
The teachings of Demetrix as applied to claims 1 and 14 are summarized above.
Demetrix is silent on the sequence of the exogenous FAD synthase that is expressed by the cell.
However, Uniprot database entry Q6C7T3 teaches a FAD synthase from Yarrowia lipolytica which provides for the biosynthesis of FAD cofactor.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the current instant application to substitute the exogenous FAD synthase taught by Demetrix with the FAD synthase taught by Uniprot database entry Q6C7T3. One of ordinary skill in the art of protein engineering would be motived to do so since Uniprot teaches another optional FAD synthase that can be used for the biosynthesis of FAD cofactor in the cell taught by Demetrix. One of ordinary skill in the art of protein engineering would have high expectations of success in doing so as Demetrix and Uniprot database entry Q6C7T3 teach the necessary methods and reagents to do so.
Therefore, claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020) as applied to claims 1, 14, and 15 above, and further in view of Uniprot Database entry (Q6C7T3; August 16, 2004).
Claims 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020) as applied to claim 1 above, and further in view of Gleeson et al. (In: Higgins, D.R., Cregg, J.M. (eds) Pichia Protocols. Methods in Molecular Biology, vol 103. Humana, Totowa, NJ., pg. 81-94; published 1998), hereinafter referred to as Gleeson.
The teachings of Demetrix as applied to claim 1 are summarized above.
Demetrix further teaches that in some embodiments, the host cell is a protease-deficient strain of Saccharomyces cerevisiae (see Paragraph 00430, pg. 144).
Demetrix does not specifically teach that the in the host cell, expression of one or more proteases are inactivated wherein the one or more proteases inactivated is selected from YALI0F099163p and homologs and orthologs thereof. However, in the teachings of Demetrix, Saccharomyces cerevisiae protease-deficient strain has functional protease homologs of YALIOF9163p that are inactivated. Table 2 of the specification of the current instant application defines YALI0F09163p as a protease that is inactivated. Demetrix also does teach that a suitable host cell for expression of exogenous terminal cannabinoid synthase also can be Yarrowia lipolytica.( see Paragraphs 00429-00430, pg. 143-144).
Gleeson teaches that in optimizing the expression of heterologous proteins, the issue of proteolysis is often an important factor, since many peptides and proteins are susceptible to degradation by proteases produced in the host organism. Gleeson teaches that even if the protein is expressed at high levels, overall yield can be drastically reduced through proteolysis. Gleeson further teaches that the use of protease-deficient strains has been shown to be a successful approach to improve the yield of fully active, expressed protein in both Saccharomyces cerevisiae and E. coli (See Abstract, pg. 81).
It would therefore have been obvious to one of ordinary skill in the art before the effective filing date of the current instant application, to inactivate protease functional homologs in the Yarrowia lipolytica as performed in the Saccharomyces cerevisiae host cell taught by Demetrix and Gleeson. One would be motivated to do so to avoid complications from proteolytic activity which may decrease the yield of expressed proteins such as the cannabinoid synthases. One of ordinary skill in the art of cloning and protein purification would have expectation of success as both Demetrix and Gleeson provide the necessary teachings and guidance.
Therefore, claims 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Demetrix (WIPO International Publication No: WO 2020/069142 A1; published April 2, 2020) as applied to claim 1 above, and further in view of Gleeson et al. (In: Higgins, D.R., Cregg, J.M. (eds) Pichia Protocols. Methods in Molecular Biology, vol 103. Humana, Totowa, NJ., pg. 81-94; published 1998).
Claims 1 and 25 are rejected under pre-AIA 35 U.S.C. 102(a)1 and 102(a)2 as anticipated by or, in the alternative, under pre-AIA 35 U.S.C. 103(a) as obvious over Genomatica, Inc. (WIPO International Publication Number WO 2021/211611 A1; published October 21, 2021, International filing date April 13, 2021), hereinafter referred to as Genomatica.
With regards to claim 1, Genomatica discloses an engineered cell for making non-natural THCAS (terminal cannabinoid synthases) (see description). Genomatica discloses that the non-natural cannabinoid synthase comprises at least one amino acid variation as compared to the wild-type cannabinoid synthase THCAS (see Abstract). Genomatica further discloses that the cells may have a second plasmid that express chaperones (overexpression) to help facilitate correct protein folding of THCAS (see paragraph 00438, pg. 155). In particular, Genomatica discloses that the chaperone expression plasmid pGro7 may be in the E. coli cells along with the plasmid that expresses the THCAS (see paragraph 00438, pg. 155).
With regards to claim 25, Genomatica discloses that in some embodiments, the engineered cell comprises a prenyltransferase (see Paragraph 0053, pg. 17) that can catalyze conversion of olivetolic acid (OA) to CGBA (see Paragraph 0070, pg. 22) and in some embodiments provides a method for making CBGVA (see Paragraph 0056, pg. 20).
It would have been obvious to one of ordinary skill in the art of protein engineering to modify the host cell taught by Genomatica to incorporate overexpression of one or more chaperones and to also incorporate the expression of a prenyltransferase as taught by Genomatica that can that can catalyze conversion of olivetolic acid (OA) to CGBA and also make CBGVA. One of ordinary skill in the art would be motivated to do so because expression of one or more chaperones would aide in protein folding and quality control and incorporation of a prenyltransferase would enable product formation such as CGBA and CBVA. One of ordinary skill in the art of protein biochemistry would have expectations of success in doing so as Genomatica teaches all the necessary methods to do so.
Therefore, claims 1 and 25 are rejected under pre-AIA 35 U.S.C. 102(a)1 and 102(a)2 as anticipated by or, in the alternative, under pre-AIA 35 U.S.C. 103(a) as obvious over Genomatica, Inc. (WIPO International Publication Number WO 2021/211611 A1; published October 21, 2021, International filing date April 13, 2021).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to GEORGE T LOUNTOS whose telephone number is (571)272-0502. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
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/GEORGE THEMISTOCLIS LOUNTOS/ Examiner, Art Unit 1652
/ROBERT B MONDESI/ Supervisory Patent Examiner, Art Unit 1652
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